Method of DNA sequencing using cleavable tags

ABSTRACT

The present invention provides novel systems for sequencing nucleic acid molecules using dNTPs that are 3′ end labeled with cleavable tags that block further extension and uniquely identify the bases to which they are attached. Removal of the tags liberates the 3′ ends of the extension products for further extension. In related embodiments, oligonucleotides containing sequence-related cleavable tags are employed in a ligation reaction to determine the sequence of a particular DNA sample.

BACKGROUND OF THE INVENTION

[0001] DNA sequencing is an important analytical technique critical togenerating genetic information from biological organisms. The increasingavailability of rapid and accurate DNA sequencing methods has madepossible the determination of the DNA sequences of entire genomes,including the human genome. DNA sequencing has revolutionized the fieldof molecular biological research. In addition, DNA sequencing has becomean important diagnostic tool in the clinic, where the rapid detection ofa single DNA base change or a few base changes can be used to detect forexample, a genetic disease or cancer.

[0002] Most current methods of DNA sequencing are based on the method ofSanger (Proc. Natl. Acad Sci. U.S.A., 74, 5463 (1977)). This methodrelies on gel electrophoresis of single stranded nucleic acid fragmentsthat are generated when a polymerization-extension reaction of a primeris terminated by incorporation of a radioactively labeleddideoxynucleotide-triphosphate. Short strands of DNA are synthesizedunder conditions that produce DNA fragments of variable length using aDNA polymerase and deoxynucleotide triphosphates (dNTP). A small amountof dideoxynucleotide triphosphates (ddNTP) is introduced into the DNAsynthesis mixture so that chain terminating ddNTPs are sometimesintegrated into a growing strand. Typically, four different extensionreactions are performed side by side, each including a small amount ofone ddNTP. Each extension reaction produces a mixture of DNA fragmentsof different lengths terminated by a known ddNTP. The ratio of ddNTPs todNTPs is chosen so that the populations of DNA fragments in any givenextension reaction includes fragments of all possible lengths (up tosome maximum) terminating with the relevant ddNTP. The nucleic acidfragments are separated by length in the gel, typically utilizing adifferent lane in a polyacrylamide gel for each of the four terminatingnucleotide bases being detected. However, such size exclusionchromatography is generally a low resolution method limited to readingshort sequences.

[0003] A variation of this method utilizes dyes rather thanradioactivity to label the ddNTPs. Different dyes are used to uniquelylabel each of the different ddNTPs (i.e., a different dye, may beassociated with each of A, G, C, and T termination) (Smith et al. andProber et al. Science 238:336-341, 1987). In the method of Smith,.fluorescent dyes are attached to the 3′ end of the dNTP converting itinto a ddNTP. The use of four different dye labels allows the entiresequencing reaction to be conducted in a single reaction vessel andresults in a more uniform signal response for the different DNAfragments. The dye-terminated dNTPs are also able to be electrophoresedin a single lane. The advent of capillary electrophoresis furtherincreased the separation efficiency of this method, allowing shorter runtimes, longer reads, and higher sensitivity.

[0004] Despite these advances, DNA sequencing methods that rely onelectrophoresis to resolve DNA fragments according to their size arelimited by the rate of the electrophoresis and the number of bases thatare detectable on the gel. In addition, real-time imaging of the gel isnot possible. Accordingly, in order to increase the speed andreliability of the sequencing reaction, great effort has been made toautomate these steps. Automated DNA sequencing machines are nowavailable that are capable of high throughput sequencing for bothgenomic sequencing and routine clinical applications. However, thesenewer techniques remain cumbersome, requiring specialized chemicals andthe intensive labor of skilled technicians.

[0005] One newer method of DNA sequencing, “pyrosequencing” or“sequencing-by-synthesis,” disclosed in WO 98/13523, is based on theconcept of detecting inorganic pyrophosphate (PPi), which is releasedduring a polymerase reaction. As in the Sanger method, a sequencingprimer is hybridized to a single stranded DNA template and incubatedwith a DNA polymerase. In addition to the polymerase, the enzymes ATPsulfurylase, luciferase, and apyrase, and the substrates, adenine 5′phosphosulfate (APS) and luciferin, are added to the reaction.Subsequently, individual nucleotides are added. When the addednucleotide is complementary to the next available base in the templatestrand, it is incorporated into the extension product. Suchincorporation of a complementary base is accompanied by release ofpyrophosphate (PPi), which is converted to ATP in the presence ofadenosine 5′ phorphosulfate by apryase in a quantity equimolar to theamount of incorporated nucleotide. The ATP generated by the reactionwith apyrase then drives the luciferase mediated conversion of luciferinto oxyluciferin, generating visible light in amounts that areproportional to the amount of ATP and thus the number of nucleotidesincorporated into the growing DNA template. The light produced by theluciferase-catalyzed reaction is detected by a charge coupled device(CCD) camera and detected as a peak in pyrogram™.

[0006] In a pyrosequencing reaction, if the first nucleotide added tothe reaction is not complementary to the next available nucleotide onthe growing DNA strand there is no light generated. If no light isgenerated by the addition of the first nucleotide, a second of fourdNTPs is added sequentially to the reaction to test whether it is thecomplementary nucleotide. This process is continued until acomplementary nucleotide is added and detected by a positive lightread-out. Whether or not a positive light reaction is generated,apyrase, a nucleotide-degrading enzyme, continuously degradesunincorporated dNTPs and excess ATP in the reaction mixture. Whendegradation is complete, another dNTP is added.

[0007] Although pyrosequencing is capable of generating high qualitydata in a relatively simple fashion, this method has several drawbacks.First, the productivity of the method is not high, reading only about 1base per 100 seconds. The rate of the reaction is limited by thenecessity of having to add new enzymes with each addition of the dNTPsin addition to the necessity of having to test each of the four dNTPsseparately. In addition, it has been found that the dATP used in thechain extension reaction interferes in subsequent luciferase-baseddetection reactions by acting as a substrate for the luciferase enzyme.Finally, these reactions are expensive to run.

[0008] While pyrosequencing improves the ease and speed with which DNAsequencing is achieved, there exists the need for improved sequencingmethods that allow more rapid detection. Preferred techniques would beamenable to automation and allow the sequence information to be revealedsimultaneously with or shortly after the chain extension reaction.

SUMMARY OF THE INVENTION

[0009] The present invention provides a novel system for sequencingnucleic acid molecules. In particular, the invention utilizes dNTPs thatare 3′ end labeled with a cleavable tag that distinguishes the dNTP fromother dNTPs (e.g., the tag may be unique to the dNTP). The cleavabletags are functional groups that can be later removed by any appropriatemeans, including but not limited to, exposure to chemical cleavageconditions or light. dNTPs labeled with the cleavable tags function asterminated dNTPs (cdNTPs), in that their incorporation into a singlestranded nucleic acid molecule via a primer extension reaction blocksfurther extension. However, removal of the tag converts the cdNTP backinto an extendible nucleotide.

[0010] According to the present methods, a sequencing primer ishybridized to a nucleic acid template, e.g., a single stranded DNAtemplate, and incubated with an enzyme (DNA polymerase) and four cdNTPs(tag terminated DATP (cdATP), dCTP (cdCTP), dGTP (cdGTP), and dTTP(cdTTP)). The DNA polymerase then extends the primer by adding to itwhichever cdNTP is complementary to the next available base on thetemplate strand. Only a single cdNTP is incorporated, because the cdNTPcannot be further extended.

[0011] After completion of a single base addition, unreacted (excess)cdNTPs are removed from the reaction mixture, which includes theextended primer, the DNA polymerase, and the single stranded DNAtemplate. The step of removing can be accomplished by any of a varietyof means that would be apparent to one skilled in the art. For example,if the reaction mixture is contained in a chamber that has an attachedmembrane (e.g., an ultrafiltration membrane that allows small moleculessuch as water, salts, and cdNTPs to pass through, but does not allowpassage of large molecules such as single stranded DNA), the excesscdNTP can be washed through the membrane. Alternatively, if the singlestranded DNA is attached to a solid support, the excess cdNTPs can bewashed away from the single stranded DNA without dislodging thehybridized, extended primer.

[0012] Once the step of removing is complete, the tag is cleaved fromthe cdNTP that is extended into the single stranded DNA template. Incertain embodiments, the cleavage occurs by photo-cleavage of the tagfrom the extended single stranded DNA template by exposure to light.Alternatively, in other preferred embodiments, the cleavage occurs byexposure of the single stranded DNA template to a chemical cleavingagent, e.g., an acid or a base. Whichever cleavage method is employed,the result is liberation of the 3′ end of the extension product forfurther extension.

[0013] The cleaved tag is then washed through the membrane into adetector for identification, thereby identifying the complementary basein the single stranded DNA template and determining the DNA sequence.The detector used to identify the tag is chosen based on the type ofcleavable tag employed. Any of a variety of tags may be employed in thepresent invention, as would be recognized by the skilled artisan, andsuch tags are described herein. Once the tag is cleaved, the four cdNTPsare added back to the primer extension reaction mixture and the cycle ofextension, tag cleavage, and identification is repeated.

[0014] In other preferred embodiments, short oligonucleotides areemployed in a ligation reaction to determine the sequence of aparticular DNA sample. The sequence of a DNA sample is determined byincorporating “X” complementary bases (e.g., 2 mers, 3 mers, or more) ata time onto the single stranded DNA template adjacent to a primer usinga DNA ligase instead of using a DNA polymerase. Each oligonucleotide istagged and labeled with a cleavable tag so that the position of eachbase in the sequence of the oligonucleotide can be identified. The tagfurther prevents ligation of the oligonucleotides to one another.

[0015] According to this aspect of the invention, a template DNA isexposed to the oligonucleotides, the oligonucleotides are allowed tohybridize to the template DNA, and a ligation reaction is allowed totake place on the DNA template such that one complementaryoligonucleotide is incorporated onto the DNA template adjacent to theannealed primer. Following ligation, the unincorporated oligonucleotidesare washed away from the DNA sample and the tags are cleaved andanalyzed to determine the nucleic acid sequence.

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS

[0016] The present invention provides a system for sequencing a DNAmolecule using deoxynucleotide triphosphates of adenine, thymine,guanine, and cytosine that are each labeled with a different cleavabletag that is used to identify the base. In preferred embodiments, thecleavable tag further acts as a terminator to extension of a singlestranded DNA template in a polymerase extension reaction until the tagis removed from the incorporated base. Once removed, the tag is isolatedand identified, and the process of base addition and cleavage isrepeated. More particularly, the steps of extension, cleavage, anddetection are repeated until sufficient sequence of the single strandedDNA template is determined.

[0017] According to certain preferred embodiments, inventive methods ofdetermining the sequence of a nucleic acid include the steps of (a)hybridizing an oligonucleotide to a single stranded DNA, wherein theoligonucleotide is complementary to at least a portion of the singlestranded DNA; (b) providing a DNA polymerase and four deoxynucleotidetriphosphates (dNTPs) (e.g., dATP, dGTP, dCTP, and dTTP) wherein eachdNTP is 3′ end labeled with a cleavable tag (cdNTP) that distinguishesthe dNTP from other dNTPs; (c) extending the single stranded DNAhybridized to the oligonucleotide by adding one complementary cdNTP in apolymerase extension reaction, wherein the tag on the extended cdNTPblocks further extension by the DNA polymerase; (d) optionally removingexcess cdNTPs that are not extended onto the single stranded DNA; (e)cleaving the tag from the extended cdNTP; and (f) detecting the tag sothat the incorporated base is detected. In certain preferredembodiments, the method includes the step of repeating steps (a) through(f) on the sample of single stranded DNA.

[0018] As indicated above, prior to cleavage of the tag from theextended base on the DNA template, the excess, unincorporated cdNTPs arepreferably removed from the extension reaction. According to theinvention, the tags may be removed by any of a variety of washing orrinsing procedures that separate the excess, unincorporated dNTPs fromthe extended DNA template. In one preferred embodiment, the extensionreaction is contained within a chamber that has an attached filtrationmembrane, e.g., an untrafiltration membrane, that allows small moleculessuch as water, salts, and cdNTPs to pass through, while retaining largemolecules such as ssDNA. According to this particular embodiment, a washsolution, e.g., a buffered saline solution such as phosphate bufferedsaline, is passed through the ultrafiltration membrane of the chambercontaining the oligonucleotide primer, the DNA polymerase, the cdNTPs,and the extended DNA to rinse away the excess cdNTPs. Alternatively, ifthe DNA template is attached to a solid support, a wash solution may bepassed over the solid support to rinse the excess cdNTPs away from thesolid support.

[0019] In a related embodiment, the sequencing method of the presentinvention is also amenable to sequence determination via oligonucleotideligation. This technique requires first exposing the DNA template to acollection of tagged oligonucleotides (e.g., the tagged oligonucleotidesmay be a collection of short randomized oligonucleotides). Preferably, a3′ tag blocks further litgation at the 3′ end of the oligonucleotide toother oligonucleotides in the collection. However, it will beappreciated that if the tag is located at a position on theoligonucleotide other than the 3′ end, the 3′ end of the oligonucleotidewould still need to be blocked, for example, with another functionalgroup. Once the DNA template is mixed with the tagged oligonucleotides,the oligonucleotides are allowed to hybridize to the DNA template in aposition adjacent to an oligonucleotide primer so that theoligonucleotide and primer can be ligated. Unligated oligonucleotidesare then rinsed away from the DNA template, tags are cleaved from theligated oligonucleotides, and cleaved tags representing the bases of theligated oligonucleotide are detected. This cycle can be repeated asdescribed, with addition of the oligonucleotide mix occurring at eachrepetition.

[0020] In certain preferred embodiments, the number of tags attached tothe 3′ end of the oligonucleotide may be based on the sequence length ofthe oligonucleotide. For example, an oligonucleotide that is three baseslong may be 3′ end labeled with three tags that are attached in asequential order matching the sequential order of the bases of theoligonucleotide.

[0021] In the oligonucleotide ligation reaction, as with the polymerasereaction, the DNA template is a single stranded DNA template that isannealed to a primer for primer extension. By “single stranded DNAtemplate” is meant any single stranded DNA template or single strandedDNA template that is partially single stranded, i.e., may be partiallydouble stranded. In one preferred embodiment, an oligonucleotide that is3′ end blocked and complementary to the sequence adjacent to the primeranneals to the DNA template and is joined to the adjacent primer via aligase (e.g., T4 DNA ligase). The tags on the complementaryoligonucleotide are then removed for detection and identification,freeing the 3′ end of the complementary oligonucleotide for subsequentrounds of ligation. In such subsequent rounds, the ligase joins the nextcomplementary blocked oligonucleotide to the 3′ end of the previouslyextended primer and the cycle repeats.

[0022] As mentioned above, the collection of oligonucleotides mayinclude short randomized oligonucleotides. Those skilled in the art willappreciate that the longer the oligonucleotide, the greater the numberof oligonucleotides will have to be generated to encompass all possiblerandom oligonucleotide sequences, based on randomization between fourbases at each position of the oligonucleotide. For example, generationof a collection of 2 mers that encompasses all possible 2 mers wouldrequire sixteen oligonucleotide sequences; generation of a collection of3 mers that encompasses all possible 3 mers would require a panel of 64oligonucleotide sequences; 4 mers would require a panel of 256oligonucleotide sequences, etc. Identification of an optimaloligonucleotide length may require simply testing various short randomoligonucleotide mixes and determining which give the most rapid andaccurate DNA sequencing results via oligonucleotide ligation. Of course,the longer the oligonucleotide, the faster the sequencing reaction willproceed, due to the increased number of incorporated bases detectedsimultaneously. Using this approach, at each round of the sequencingreaction, the oligonucleotide sequence that is ligated to the primer isdetected and identified.

[0023] In certain preferred embodiments, it is conceivable that only asmall subset of all possible oligonucleotides need to be used in thesequencing reaction, for example, if the sequence of the DNA templatewere partially determined (i.e., if certain positions of theoligonucleotide were fixed, fewer base positions would need to berandomized, limiting the number of oligonucleotide required to includeall possible permutations). In this particular embodiment, theoligonucleotides could be longer (e.g., 5 mer, 6 mers, 7 mers, 8 mers, 9mers, 10 mers, or greater than 15 mers, 20 mers, 25 mers, 30 mers andhigher). It is also possible that in certain circumstances one would notneed to use as many tags i.e., one would not need to use one tag forevery base. For example, one unique tag could be used to identify anentire oligonucleotide sequence.

[0024] In other preferred embodiments, two or more unique tags could beused to identify an entire oligonucleotide sequence, the total number oftags being less than the total number of bases in the oligonucleotides(e.g., each tag could identify short sequential stretches ofoligonucleotides (e.g., a 3 mer or a 4 mer etc.) within the entireoligonucleotide sequence). In a related embodiment, an oligonucleotide,particularly an oligonucleotide used in the ligation aspect of theinvention, may not be randomized at every position (e.g., if certainnucleotide positions are fixed), and may even be randomized at only oneor several positions, e.g., 1-2, 1-3, 1-4 or 1-5 positions. Under thesecircumstances, only a subset of possible variations would be relevant.

[0025] In embodiments where the length of the oligonucleotide sequenceincreases the number of tags required to identify the oligonucleotidesequence, the availability of many unique mass tags makes massspectrometry a particularly useful system for detection. Since eachshort random oligonucleotide must be labeled with a unique tags, theshort random oligonucleotide may have a maximum length in certaincircumstance (e.g., the length and number of oligonucleotides in acollection of oligonucleotides may be limited by the availability ofdifferent unique tags). However, mass tags may have the same nominalmass and vary in structure, thereby increasing the diversity of tagsavailable.

[0026] Although the level of diversity available in the massspectrometry system is sufficient to permit unique MS/MS fragmentation,those skilled in the art will appreciate that, because identification ofthe incorporated oligonucleotides is based on the MS/MS parent/daughtertransition, if an MS/MS approach is used, multiplexing target DNAsamples is not possible. The MS/MS approach requires the isolation of asingle mass followed by fragmentation and mass analysis. Multiplexingwould present too many masses for isolation and fragmentation to bepractical. However, the MS/MS approach would be helpful in increasingthe potential number of mass tags required for coding theoligonucleotides used in the ligation reaction.

[0027] Thus, according to the oligonucleotide ligation aspect of theinvention, the sequence of a single stranded DNA template may bedetermined by (a) hybridizing a complementary oligonucleotide to asingle stranded DNA adjacent to a primer, wherein the oligonucleotide is3′ end labeled with one or more cleavable tags unique to theoligonucleotide sequence; (b) ligating the hybridized complementaryoligonucleotide to the primer, wherein the one or more tags on theextended cdNTP blocks further ligation by the DNA ligase; (c) optionallyremoving excess oligonucleotides that are not ligated; (d) cleaving theone or more tags from the ligated complementary oligonucleotide; (e)detecting the one or more tags. In certain preferred embodiments, steps(a) through (e) are repeated on the single stranded DNA.

[0028] As in the polymerase reaction, in the ligation reaction, prior tocleavage of the tag(s) from the extended oligonucleotide on the DNAtemplate, the excess oligonucleotides are preferably removed. Theoligonucleotides may be removed by any of a variety of separationprocedures that may include washing or rinsing the unincorporatedoligonucleotides away from the extended DNA template. As with thepolymerase reaction, in one preferred embodiment, the ligation reactionis contained within a chamber that has an attached filtration membranethat would allow short oligonucleotides to pass through, while retaininglarger molecules, such as the DNA template. Alternatively, the DNAtemplate is attached to a solid support and a wash solution may bepassed over the solid support to remove the unincorporated taggedoligonucleotides.

[0029] As will be appreciated by those skilled in the art, whether thesequencing reaction employs a DNA polymerase or a DNA ligase, any tagthat is cleavable by chemical means or by light can be used in thepresent invention. In certain preferred embodiments, the tag is cleavedby exposure to an acid or a base. In other preferred embodiments. thetag is cleaved by exposure to light, i.e., in a photo-cleavage reaction.The cleavable tags themselves include any functional group that impartsa unique identity onto the oligonucleotide or base that is tagged.According to the present invention, useful tags include, e.g.,fluorescent tags, mass tags, IR tags, UV tags, potentiometric tags, etc.For example, a fluorescent tag may be attached to a dNTP prior to theprimer extension reaction, and then may be cleaved from the dNTP afterthe dNTP is incorporated into the extended DNA strand by exposure of theextended DNA strand to an acid, a base, or light, and analyzed usingfluorescence spectrometry. As but another example, a base having anacid, base, or light cleavable mass tag, after incorporation into theDNA template, may be cleaved from the extended DNA strand using theappropriate cleavable agent, and then may be analyzed using massspectrometry.

[0030] The DNA sequencing methods of the present invention provide anadvantage over existing Sanger-based methods by eliminating the need toseparate cDNA fragments on a gel, resulting in longer sequence reads.The present method is rapid and fully automatable. In addition, theselection and detection of one of the four bases is carried outsimultaneously.

[0031] Alternatively, the identification step need not be carried outsimultaneously with the cycling of the reaction. For example, the tagsfrom each cycle may be collected and pooled (e.g., onto a 96 wellplate). Alternatively, the tags from each cycle may be spatially arrayed(e.g., onto a chip) and the positional information used foridentification. Using either method, the tags are analyzed subsequent tothe cycling reaction by art available means. Such collection andanalysis may increase the speed of the sequencing reaction to increasethe throughput of the technique. Of course one skilled in the art wouldrecognize that the appropriate instrumentation is required to analyzethe collected tags.

[0032] Certain aspects of the present invention are described in furtherdetail below.

[0033] Nucleic Acid Preparation

[0034] In certain preferred embodiments of the invention, the DNA sampleis a single stranded DNA template. Alternatively, if in a polymeraseextension reaction a thermostable DNA polymerase enzyme is employed, theDNA sample may be double stranded.

[0035] The DNA sample of the invention may be provided from anyavailable source of DNA, including, for example, a biological sample,including not only samples obtained from living organisms (e.g.,mammals, fish, bacteria, parasites, viruses, fungi, and the like) orfrom the environment (e.g., air water, or solid samples), but biologicalmaterials which may be artificially or synthetically produced (e.g.,phage libraries, organic molecule libraries, pools of genomic clones,and the like). Representative examples of biological samples includebiological fluids (e.g., blood, semen, cerebral spinal fluid, urine),biological cells (e.g., stem cells, B or T cells, fibroblasts, and thelike), and biological tissues. Alternatively, the DNA may be a cDNAsynthesized from an RNA sample (e.g., from a natural or syntheticsource). Such cDNA synthesis may be carried out using reversetranscription, and such systems are readily available.

[0036] The DNA sample, whether from a biological or synthetic source,may further be amplified, particularly if the amount of sample DNA issmall. Amplification can be carried out by any art available method, forexample, in vitro by PCR or Self Sustained Sequence Replication (3SR) orin vivo using a vector. Alternatively, if desired, in vitro and in vivaamplification may be used in combination (see, e.g., McPherson, “PCR. APractical Approach,” Oxford University Press, New York, 1991). Withinother embodiments of the invention, the DNA samples of the presentinvention may be generated by, for example, a ligation or cleavagereaction.

[0037] According to the invention, the DNA sample, amplified orunamplified, is either immobilized on a solid support or in solution. Inthe case of an amplified DNA sample, those skilled in the art willrecognize that any amplification procedure may be modified to allow forattachment of the amplified DNA sample to a solid support. For example,a chosen PCR primer may be immobilized to a solid support or may beprovided with a means for attachment to a solid support. Immobilizationmay take place as part of a PCR amplification, e.g., where one or moreprimers is attached to a support. Alternatively, one or more primers maycarry a functional group, e.g., a biotin or thiol group, permittingsubsequent immobilization of the DNA sample. Immobilization of the 5′end of a DNA in the sample, e.g., via a 5′ primer, allows the DNA to beattached to a solid support, leaving its 3′ end remote from the supportand available for subsequent hybridization with the extension primer andextension by the polymerase (or ligase). Alternatively, an unamplifiedDNA sample, such as a vector or a biological sample, may include, or bemodified to include, a functional group that allows attachment to asolid support. In a related embodiment, the vector may include a meansfor attachment to a solid support adjacent to the site of insertion ofthe sample DNA such that the amplified DNA sample and the means forattachment may be excised together.

[0038] The solid support may conveniently take the form of, for example,microtiter wells, a solid support activated with polystyrene to bind theDNA sample (e.g., primer DNA), particles, heads. (e.g., nylon beads,polystyrene microbeads, or glass beads) (Polysciences, Warrington, Pa.),glass surfaces, plates, dishes, flasks (Corning Glass Works, Corning,N.Y.), meshes (Bectorn Dickinson, Mountain View Calif.), membranes(Millipore Corp., Bedford, Mass.), dipsticks, capillaries, hollow fibers(Amicon Corporation, Danvers, Mass.), screens and solid fibers (Edelmanet al., U.S. Pat. No. 3,843,324; see also Kuroda et. al., U.S. Pat. No.4,416,777, incorporated herein by reference), or needles, made, forexample, of agarose, cellulose, alginate, Teflon, or polystyrene.Magnetic particles, e.g., majestic beads, may also be used as solidsupports, and such materials are commercially available (RobbinScientific, Mountain View, Calif.).

[0039] The solid support may alternatively or additionally carryfunctional groups such as hydroxyl, carboxyl, aldehyde, or amino groups,or other moieties, such as avidin or streptavidin, for the attachment ofthe appropriately modified DNA, e.g., via modified oligonucleotideprimers used in an amplification reaction. These may in general beprovided by treating the support to provide a surface coating of apolymer carrying one of such functional groups, e.g., polyurethanetogether with a polyglycol to provide hydroxyl groups, or a cellulosederivative to provide hydroxyl groups, a polymer or copolymer of acrylicacid or methacrylic acid to provide carboxyl groups, or anaminoalkylated polymer to provide amino groups. Various other supportsand methods of attachment and detachment of nucleic acid molecules tosupports, with and without the use of a linker, is described in U.S.Pat. No. 5,789,172, incorporated herein by reference.

[0040] As indicated above, the DNA sample need not be attached to asolid support. For example, a polymerase extension reaction may becarried out in solution on a DNA sample that is prepared in the contextof a primer extension reaction having a buffer that will accommodate theaddition of an oligonucleotide primer, a DNA polymerase, cdNTPs, and asingle or double-stranded DNA template. A ligation extension reactionmay be similarly carried out in an appropriate buffer in the presence ofan oligonucleotide primer, a DNA ligase, tagged oligonucleotide, and asingle or double-stranded DNA template.

[0041] Extension

[0042] Once a suitable DNA sample is prepared, the sample is subject toa primer extension reaction by addition of an oligonucleotide primer, aDNA polymerase, and four cdNTPs, such that one base is incorporated ontothe DNA template before extension is blocked by the cleavable tag on theincorporated base. Alternatively, an oligonucleotide ligation reactionis used to extend the template DNA sample, as described above. Thoseskilled in the art will appreciate that such extension reactions can bemodified to accommodate variations in template DNAs, reactionconditions, etc. It will be further recognized that the chosenoligonucleotide primer must be sufficiently large to provide appropriatehybridization with the target DNA sequence. Moreover, theoligonucleotide primer preferably hybridizes immediately 5′ to thetarget sequence. Guidance for selection of primers and primer extensionreactions can be found in the scientific literature, for example,Maniatis et al., Molecular Cloning, a laboratory Manual (1989).

[0043] The polymerase in the primer extension reaction may be anypolymerase that incorporates dNTPs, and preferably cdNTPs, onto a singlestranded DNA template. Examples of suitable polymerases that mayconveniently be used, and many are known in the art and reported in theliterature, include T7 polymerase, Klenow, and Sequenase. Those skilledin the art will be aware that certain polymerases, e.g., T7 polymerase,recognize a specific leader sequence in the DNA, which can be includedin the sequence of the oligonucleotide primer. If a double stranded DNAtemplate is to be used in the polymerase extension reaction, it isdesirable that a thermostable polymerase, such as a Taq polymerase, bechosen to permit repeated temperature cycling without having to addadditional polymerase for each round of extension.

[0044] It is well known that many polymerases have a proof-reading orerror checking ability, which sometimes results in digestion of 3′ endsavailable for extension. In the method of the invention, such digestionmay result in an increased level of background noise. In order to avoidthis problem, a nonproof-reading polymerase, e.g., an exonucleasedeficient (exo-) Klenow polymerase may be used. Otherwise, fluoride ionsor nucleotide monophosphates that suppress 3′ digestion by thepolymerase may be added to the extension reaction mixture. In addition,it may be advantageous to use an excess amount of polymerase overprimer/template to maximize the number of free 3′ ends that areextended. Those skilled in the art will appreciate that the precisereaction conditions and concentrations of reactants etc. may readily bedetermined for each system according to choice.

[0045] Since the primer is extended by a single base (or a singleoligonucleotide) by the methods described above, the extended primerserves in exactly the sane way in the repeated procedure, and with eachsubsequence base (or oligonucleotide) addition, to determine the nextbase or bases in the sequence, permitting the whole sample to besequenced.

[0046] Separation

[0047] In the case of the polymerase extension reaction, prior tocleavage of the tag from the extended DNA template, the excess cdNTPsmust be removed from the reaction mixture to prevent contamination ofthe cleavage product with signals from other unincorporated bases. Asmentioned above, this separation may be accomplished by washing thecdNTPs through a membrane filter that allows flow through of smallmolecules such as water, salts, and cdNTPS, but does not allow the flowthrough of larger molecules such as the polymerase and the DNA template.

[0048] In the case of the ligase extension reaction, prior to cleavageof the tag from the oligonucleotide on the extended DNA template, theunincorporated tagged oligonucleotide must be removed from the reactionmixture to prevent contamination of the cleavage product with signalsfrom the unincorporated tagged oligonucleotides. Depending on whetherthe DNA template is free in solution or attached to a solid support, theexcess unincorporated tagged oligonucleotide may be removed by eitherfiltration or washing the solid support, respectively. Those skilled inthe art will appreciate that if the DNA ligase is removed from theextension reaction mixture along with the tagged oligonucleotide, theligase will need to be added back to the extension reaction mixture insubsequent rounds. This, of course, is also applicable to a sequencingreaction that utilizes a polymerase, where the polymerase is removedfrom the extension reaction in a separate step with the cdNTPs.

[0049] Those skilled in the art will further appreciate that a widevariety of membrane filters are available in the art. For example,molecular filtration, also known as ultrafiltration, is a membraneseparation technique used to segregate substances according to molecularweight and size. Molecular filtration is ideally suited to separatesalts and other low molecular weight solutes from high molecular weightspecies. Molecular filtration-is based on a pressure differential acrossthe semipermeable membrane to drive permeable materials through themembrane. For this reason, molecular filtration typically separatessolutes and concentrates retained materials more rapidly. Molecularfiltration membranes appropriate for use in the present invention may bepurchased from Millipore Corp., Bedford, Mass.

[0050] In another preferred embodiment, a flow through cell is used forsingle stranded DNA analysis. In this embodiment, the tag is washed awayand is sent to the detector directly. One example of a variation on theflow through cell approach that would be amenable to multiplexing is touse a 96 well plate with an ultrafiltration membrane incorporated in thewell. The excess reagents are either washed through by pressure orcentrifuged through. The tag is then subsequently cleaved from extendednucleotide base, washed through the membrane, and collected for analysisby the method appropriate for the type of tag to be identified. Incertain preferred embodiments, the different wells are pooled and thetags analyzed simultaneously to provide greater sample multiplexing aswell as throughput.

[0051] Where the DNA template is immobilized on a solid support, theseparation is accomplished by simply washing the cdNTPs (or taggedoligonucleotide) away from the solid support. For example, one basicapproach to retaining the DNA for analysis would be to absorb the targetDNA to an adsorptive surface instead of trapping it behind anultrafiltration membrane. The excess reagents are washed away from theabsorbed DNA by rinsing the absorptive surface with a wash solution. Thesolvents used in the wash step must be chosen to avoid loss of the DNAduring the wash steps.

[0052] The basic concept of using a membrane to permit flow of theexcess reagent away from the DNA in the wash step can be furthercombined with the concept of adsorbing the DNA to a surface byincorporating a membrane onto a microfluidic chip. Solvent addition, orwashes, may be carried out by the use of electro-osmotic flow. In thisparticular embodiment, all of the reactions and sample pooling occurs onthe chip, permitting high throughput at a lower cost compared to thewell plate approach. Within further embodiments, the steps of removing,cleaving, and detecting may be performed in a continuous manner (e.g.,as a continuous flow), for example, on a single device which may beautomated.

[0053] Cleavable Tags and Detection

[0054] A “tag,” according to the present invention, is a chemical moietythat is used to uniquely identify a nucleic acid molecule. In certainpreferred embodiments, the nucleic acid molecule is a nucleotide base.In other preferred embodiments, the nucleic acid molecule is a nucleicacid fragment, such as a DNA or an RNA. “Tag” more specifically refersto the tag variable component as well as whatever may be bonded mostclosely to it.

[0055] The tags of the present invention further possess one or more ofcertain characteristic attributes. The tag is preferably distinguishablefrom all other tags, particularly from other tags used in a particularreaction. The discrimination from other chemical moieties can be basedon the chromatographic behavior of the tag (particularly after thecleavage reaction), its spectroscopic or potentiometric properties, orsome combination thereof. In addition, the tag is capable of beingdetected when present at 10⁻²² to 10⁻⁶ mole. The tag is furtherattachable to the nucleic acid molecule, e.g., nucleotide base oroligonucleotide, through a “chemical handle” (see U.S. Pat. No.6,027,890, incorporated herein by reference) which may attach the tag tothe nucleic acid molecule either directly, or through a linker group. Incertain preferred embodiments, the tags block primer extension. The tagsare further stable toward all manipulations to which they are subjected,including attachment to the nucleic acid molecule and cleavage from thenucleic acid molecule, and any manipulations of the nucleic acidmolecule while the tag is attached to it; nor does the tag significantlyinterfere with the manipulations performed (e.g., hybridization orenzymatic reactions) on the nucleic acid molecule while the tag isattached to it.

[0056] The tags of the present invention include any tag that iscleavable by chemical means or by light, and such tags are discussed indetail below. Chemically cleavable tags include tags that are cleavableby an acid or a base. Photo-cleavable tags include tags that arecleavable by a wavelength of light. Other methods of cleavage includeoxidation, reduction, enzymatic, electrochemical, heat, and the like.

[0057] As mentioned above, the tag is further capable of terminating aprimer extension reaction. In certain preferred embodiments, theterminating nature of the tag may be due to the nature of the tagitself, for example the structure of the tag, e.g., a tag that issufficiently bulky in its structure so that that it prevents addition ofany additional bases to the extension product. Alternatively oradditionally, the terminating nature of the tag may be due to theplacement of the tag on the base. Preferably, the tag is attached to thebase so that when the base is added to the growing 3′ end of theextension product the tag effectively blocks the extension of the 3′ endby additional bases once a tagged base has been added. One such exampleof a tagged base, wherein the tag is attached directly to the base, thatwould block extension is shown below.

[0058] Alternatively, the tag is linked via a labile bond (or labilebonds) to the 3′ position of the dNTP, as shown below,

[0059] wherein:

[0060] L is the linker.

[0061] According to the invention, the tag, including the linker incases where a linker is employed, or other 3′ blocking group, areremoved to expose the 3′ hydroxyl group of the base. Exemplary tags andlinkers are described in detail in U.S. Pat. No. 6,027,890, incorporatedherein by reference.

[0062] In light of the availability of numerous tags, any number of tagsmay be utilized in a given reaction simultaneously, or within differentreactions in an array. In certain preferred embodiments, particularlywith respect to detection of ligation products, as described below, 4,5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200,250, 300, 350, 400, 450, or greater than 500 different and unique taggedmolecules may be utilized within a given reaction simultaneously,wherein each tag is unique for a selected base, oligonucleotide, orother nucleic acid fragment.

[0063] The characteristics of a variety of well known tags that areamenable to attachment to the bases and nucleic acid molecules of theinvention are described in U.S. Pat. No. 6,027,890, incorporated hereinby reference. Such tags are detectable, once cleaved from the extendedbase, by fluorometry, mass spectrometry (MS), infrared (IR)spectrometry, ultraviolet (UV) spectrometry, or potentiostaticamperometry (e.g., utilizing coulometric or amperometric detectors).Mass spectrometry is particularly amendable to multiplexing with massdetection. Representative examples of suitable mass spectrometrictechniques include time-of-flight mass spectrometry, quadrupole massspectrometry, magnetic sector mass spectrometry, and electric sectormass spectrometry. Specific embodiments of such techniques includeion-trap mass spectrometry, electrospray ionization mass specrometry,ion-spray mass spectrometry, liquid ionization mass spectrometry,atmospheric pressure ionization mass spectrometry, electron ionizationmass spectrometry, fast atom bombard ionization mass spectrometry, MALDImass spectrometry, photoionization time-of-flight mass spectrometry,laser droplet mass spectrometry, MALDI-TOF mass spectrometry, APCI massspectrometry, nano-spray mass specrometry, nebulised spray ionizationmass spectrometry, chemical ionization mass spectrometry, resonanceionization mass spectrometry, secondary ionization mass spectrometry,and thermospray mass spectrometry.

[0064] The following is a list of representative vendors for separationand detection technologies that may be used in the present invention.Perkin Elmer/Applied Biosystems Division (ABI, Foster City, Calif.)manufacturers semi-automated sequencers based on fluorescent-dyes(ABI373) and (ABI377). Analytical Spectral Devices (Boulder, Colo.)manufactures UV spectrometers. Hitachi Instruments (Tokyo, Japan)manufactures Atomic Absorption spectrometers, Fluorescencespectrometers, LC and GC Mass Spectrometers, NMR spectrometers, andUV-VIS Spectrometers. Perseptive Biosystems (Framingham, Mass.) producesMass Spectrometers (Voyager™ Elite). Bruker Instruments Inc. (ManningPark, Mass.) manufactures FTIR Spectrometers (Vector 22), FT-RamanSpectrometers, Time of Flight Mass Spectrometers (Reflex II™), Ion TrapMass Spectrometer (Esquire™) and a MALDI Mass Spectrometer. AnalyticalTechnology Inc. (ATI, Boston, Mass.) makes UV detectors and Diode ArrayDetectors. Teledyne Electronic Technologies (Mountain View, Calif.)manufactures an Ion Trap Mass Spectrometer 3DQ Discovery™ and the 3dQApogee™). Perkin Elmer/Applied Biosystems Division, (Foster City,Calif.) manufactures a Sciex Mass Spectrometer (triple quadrupoleLC/MS/MS, the API 100/300), which is cornpatible with electrospray.Hewlett-Packard (Santa Clara, Calif.) produces Mass Selective Detectors(HP 5972A), MALDI-TOF Mass Spectrometers (HP G2025A), Diode ArrayDetectors, CE units, HPLC units (HP1090), as well as UV Spectrometers.Finnigan Corporation (San Jose, Calif.) manufactures mass Spectrometers(magnetic sector and four other related mass spectrometers). Rainin(Emeryville, Calif.) manufactures HPLC instruments.

[0065] Those skilled in the art will recognize how to apply such devicesto the methods of the present invention. Those skilled in the art willfurther appreciate that devices used to detect pyrosequencing reactionsmay be adapted to detect and identify the cleaved tags of the invention.For example, the reaction monitoring system described in WO 99/66131,the microfluidic device described in WO 00/40750, the liquid dispensingapparatus described in WO 00/56455, the solid support apparatus of U.S.Pat. No. 5,302,509, each of which is incorporated herein by-reference,may be adopted for use with the method of the present invention.

[0066] Automation and High-throughput Sequencing

[0067] The DNA sequencing methods of the present invention are fullyautomatable. Those skilled in the art will recognize that the use of arobot apparatus, where a large number of samples may be rapidlyanalyzed, may be used for rapid detection and quantification of the tagmolecules. Tags to be detected spectrophotometrically may be detected,e.g., by mass spectrometry or fluorescence spectrometry. The use ofluminometers, mass spectrometers, and other spectrophotometric devicesare well known in the art and described in the literature. The DNAsequencing method of the present invention thus provides an automatedapproach for high-throughput, non-electrophoretic sequencing proceduresthat allows for continuous measurement of the progress of thepolymerization reaction in real time.

[0068] In related embodiments, it will be appreciated that multiplesamples may be handled in parallel and such parallel handling providesanother advantage to the inventive method. In order to obtain highthroughput sequence readout, multiple DNA sequencing reactions can beprocessed in parallel. According to this particular embodiment, the DNAsequencing method of the present invention can be carried out in any ofa variety of array formats.

[0069] For example, a single sequencing reaction of the invention,carried out in a single well and analyzed using flow injection analysis(FIA) has a rate of about one base every six seconds (equivalent toabout ten bases per minute and about 600 bases per hour). In order toincrease this rate, the DNA sequencing reactions may be multiplexed. Forexample, multiplexing 25 sequences increases the rate of sequencing toabout15000 bases per hour. Those skilled in the art will recognize thepower of multiplexing as it is applicable to any means of detectiondescribed herein. The number of DNA samples that can be multiplexed forparallel analysis can range 10 to 100, in some cases 100-500, and in yetsome other cases, 100-1000 or more DNA samples.

[0070] In certain preferred embodiments, an array format is used foranalysis wherein the DNA samples are distributed over a surface, forexample, a microfabricated chip, thereby immobilizing an ordered set ofsamples in a 2-dimensional format. This allows the analysis of manysamples in parallel. According to this embodiment of the invention, theDNA samples are arrayed onto any of a variety available microchips priorto commencing the sequencing reaction. Methods of producing andanalyzing DNA arrays are well known in the art and are provided in U.S.Pat. No. 6,027,789, incorporated herein by reference.

[0071] For example, applying the method of the invention to the arrayformat, after primer extension, the tags may be cleaved from the DNAsamples on the chip and pooled for analysis using spectrometric orpotentiometric techniques (e.g., MALDI-MS). In one particular embodimentof the present invention, an array interrogation system is provided thatincludes a DNA array generating device, a washing device, a tag cleavingdevice, a detecting device, and a data processor and analyzer thatanalyzes data from the detecting device to correlate a tag with anucleic acid fragment from a sample, as described in U.S. Pat. No.6,027,789, incorporated herein by reference. The arrayed DNA chip has onits surface selected DNA samples of nucleic acid fragments and cleavabletags, e.g., cleavable mass spectrometer tags, attached to the nucleicacid fragments. The arranged DNA chip is passed through or past aphotolytic cleavage device that cleaves the tags from the nucleic acidfragments while still on the DNA chip.

[0072] After the tags are cleaved, the DNA chip is positioned in anautomated micro-array sampling laser device, such as a Matrix AssistedLaser Desorption Ionization (MALDI) instrument. The MALDI instrument isadapted to irradiate and cause desorption of the tags, which aretransferred to a detection device, such as a mass spectrometer, whereintags are identified based upon the difference in molecular weight.

[0073] Data from the detection device is provided to the data processorand analyzer, which includes a software program that maps the signatureof a given tag to a specific sample. The software is able to display theDNA sequence determined and load the sequence information intorespective data bases.

[0074] In an alternative embodiment, the MALDI instrument includes anadditional light source that is capable of irradiating the entire DNAchip at a wavelength in the range of 250-360 nm with adjustableintensity, so as to cause the photolytic cleaving of the tags.Accordingly, the cleaving device is incorporated as a component of theMALDI instrument. After cleaving the tags, the MALDI instrumentvolatized the tags, which are transferred to the detecting device asdiscussed above.

[0075] In yet another embodiment the DNA chip is moved from the DNAarray generating device directly to the MALDI instrument. The MALDIinstrument includes a laser that emits at a wavelength in the range ofapproximately 250 to 360 nm, inclusive. The laser causes thesimultaneous photolytic cleavage of the tag from the nucleic acidfragment along with simultaneous desorption of the tag. The tags arethen transferred to the mass spectrometer or other detection device, asdiscussed above. Accordingly, this alternate embodiment providesphotocleavage by the MALDI instrument, so that a separate cleavagedevice is not needed.

[0076] If fluorescence sensing is employed in the present invention fordetection of the tag, this increases the rate of the sequencing to onebase every fifteen seconds (equivalent to about four bases per minute).If 100 sequencing reactions are arranged onto 100 lanes of the chip thisyields a rate of about 24000 bases per hour. Similar sequencing ratesare achievable with varying cleavage means.

[0077] Florescent tags can be identified and quantitated most directlyby their absorption and fluorescence emission wavelengths andintensities. While a conventional spectrofluorometer is extremelyflexible, providing continuous ranges of excitation and emissionwavelengths (I_(EX), I_(S1), I_(S2)), more specialized instruments, suchas flow cytometers and laser-scanning microscopes require probes thatare excitable at a single fixed wavelength. In contemporary instruments,this is usually the 488-nm line or the argon laser.

[0078] Radioactive tags may also be applicable to the present invention.Radioactive tags may be detected by,. e.g., a CCD detector.

[0079] In using fluorescent and radioactive tags, the number ofdifferent reactions that are simultaneously detectable may be morelimited than, e.g., mass tags. For example, the use of four fluorescentmolecules, such as commonly employed in DNA sequence analysis, limitsanalysis to four samples at a time.

[0080] In certain preferred embodiments, the sample reactions may bepooled on at least one array and the products detected simultaneously.By using a cleavable tag, such as the ones described herein, having adifferent molecular weight or other physical attribute in each reaction,the entire set of reaction products can be harvested together andanalyzed.

[0081] Applications

[0082] The invention in the above embodiments provides a simple andrapid method for sequencing a DNA sample. The methods of the inventionboth avoid the requirement of separation of the extension product andallows rapid, real-time analysis of the extension reaction. Thesemethods have many applications, which will readily be appreciated by theskilled artisan.

[0083] To name but a few, the present invention is applicable in thefield of forensics (e.g., the identification of individuals and thelevel of DNA sequence variations); tumor diagnosis (e.g., for detectionof viral or cellular oncogenes in a biological sample from a patient);transplantation analyses (e.g., the identification of antigen specificvariable DNA sequences from a biological sample); diagnosis ofautoimmune diseases, such as juvenile diabetes, arteriosclerosis,multiple sclerosis, rheumatoid arthritis, and encephalomyelitis; genomediagnostics (e.g., the identification of genetic defects or hereditaryand acquired genetic diseases in newborns and adults, for example,schizophrenia, manic depression, epilepsy, sickle-cell, anemia,thalessemias, a1-antitrypsin deficiency, Lesch-Nyhan syndrome, cysticfibrosis, Duchenn/Becker muscular deficiency, Alzheimer's disease,X-chromosome-dependent mental deficiency, and Huntingtins chorea);infectious disease (e.g. detection of viral or microbial infection of abiological sample); mutation detection (e.g., detection of a mutatedbase in a DNA sample from a biological or artificial source); detectionof single nucleotide changes (e.g., a primer hybridizes to a sequenceadjacent to a known single nucleotide polymorphism and a cdNTP added tothe adjacent position is detected and identified).

[0084] As mentioned above, the method of the present invention may beadapted for use with a ligase instead of a polymerase. One adaptation ofthis technique is to the oligonucleotide ligation assay, which is usedto identify known sequences in very large and complex genomes. Toelaborate briefly on the ligase extension reactions described above, thebasis of this assay is the ability of a ligase to covalently join twodiagnostic oligonucleotides as they hybridize adjacent to one another ona given DNA target. If the sequences at the probe junctions are notperfectly base-paired, the probes will not be joined by the ligase. Whentags are used, they are attached to the oligonucleotide, which isligated to the DNA sample. After a ligation is complete, the tag iscleaved and detected by any of the means described herein (e.g., massspecrometry, infrared spectrophotometry, potentiostatic amperometry, orUV/visible spectrophotometry).

[0085] In certain preferred embodiment, the DNA sample is amplifiedprior to exposure to the oligonucleotide ligation assay.

[0086] Kits

[0087] The present invention further provides kits for use in methods ofthe invention that contain at least the following reagents: a) anoligonucleotide primer suitable for primer extension of a particular DNAtemplate; b) four cdNTPs of adenine, guanine, thymine, and cytosinebases; c) a polymerase; d) a separation means to separate unincorporateddNTPS from the extended DNA template; and e) a cleavage means. Incertain embodiments of the invention, a detection means will beprovided. However, the detection means may often be provided by thepurchaser.

[0088] In alternative embodiments, if the kit is used for a ligationsequencing reaction assay it may contain at least a) an oligonucleotideprimer suitable for primer extension of a particular DNA template; b) atleast one tagged oligonucleotide; c) a ligase; d) a separation means toseparate unincorporated oligonucleotides from the extended DNA template;and e) a cleavage means. The kit may further provide a detection means.However, the detection means may also be provided by the purchaser.

[0089] Other embodiments of the invention will be apparent to thoseskilled in the art from a consideration of the specification or practiceof the invention disclosed herein. It is intended that the specificationbe considered as exemplary only, with the true scope and spirit of theinvention being indicated by the following claims.

What is claimed is:
 1. A method of determining the sequence of a nucleic acid, comprising steps of: (a) hybridizing an oligonucleotide to a single stranded DNA, wherein the oligonucleotide is complementary to at least a portion of the single stranded DNA; (b) providing a DNA polymerase and four deoxynucleotide triphosphates (dNTPs) comprising dATP, dGTP, dCTP, and dTTP, wherein each dNTP is 3′-end labeled with a cleavable tag (cdNTP) that distinguishes it from the other cdNTPs; (c) extending the single stranded DNA hybridized to the oligonucleotide by one complementary end-labeled cdNTP in a polymerase extension reaction, wherein the tag on the extended cdNTP blocks further extension by the DNA polymerase; (d) cleaving the tag from the complementary cdNTP; and (e) detecting the tag, thereby identifying the complementary dNTP.
 2. The method of claim 1, further comprising the step of removing excess cdNTPs that are not extended onto the single stranded DNA.
 3. The method of claim 1, further comprising the step of repeating steps (a) through (e) on the sample of single stranded DNA.
 4. The method of claim 1, wherein the cleavable tags are cleavable by chemical cleavage.
 5. The method of claim 4, wherein the cleavable tags are acid cleavable tags.
 6. The method of claim 4, wherein the cleavable tags are base cleavable.
 7. The method of claim 1, wherein the tags are photocleavable.
 8. The method of claim 1, wherein the tag is a fluorescent tag.
 9. The method of claim 1, wherein the tag is a mass tag.
 10. A method of determining the sequence of a nucleic acid, comprising steps of: (a) hybridizing a complementary oligonucleotide to a single stranded DNA, wherein the oligonucleotide is 3′-end labeled with one or more cleavable tags that distinguishes it from other oligonucleotides; (b) cleaving the one or more tags from the ligated complementary oligonucleotide, and (c) detecting the one or more tags.
 11. The method of claim 10, wherein the hybridizing of the complementary oligonucleotide occurs adjacent to a primer.
 12. The method of claim 11, further comprising ligating the hybridized oligonucleotide to the primer.
 13. The method of claim 12, further comprising the step of removing excess oligonucleotides that are not ligated onto the single stranded DNA.
 14. The method of claim 12, further comprising the step of repeating steps (a) through (c) on the single stranded DNA.
 15. The method of claim 10, wherein one or more tags are cleaved by chemical cleavage.
 16. The method of claim 15, wherein the cleavable tags are acid cleavable tags.
 17. The method of claim 15, wherein the cleavable tags are base cleavable.
 18. The method of claim 10, wherein the tags are photocleavable.
 19. The method of claim 10, wherein the tag is a fluorescent tag.
 20. The method of claim 10, wherein the tag is a mass tag. 